I have just synthesized first strand cDNA samples from total RNA that I would like to use as templates in RACE PCR. The concentration of the cDNA is rather high though; I have four different samples of cDNA that range from 1,100 ng/uL to 1,800 ng/uL.
My protocol requires 2.5uL of template in the 50uL reaction.

1. What is the recommended concentration of cDNA I should use as my template?

2. How do I go about diluting my samples (calculation? water vs. buffer?) to obtain the desired concentration?

THANKS!!!

jonoave

03-17-2012 10:45 AM

Re: concentration of cDNA template for PCR

As a general rule, the concentration of cDNA will vary greatly if checked using Nanodrop due to the lingering presence of other chemicals, e.g. RNA, dNTP, buffer etc. So it's not a "pure" cDNA sample, as compared to an RNA sample which you purified.

Secondly, the concentration of cDNA is generally high enough for good amplification of most genes through regular PCR. I'd say 1 ul of cDNA template in a 50 ul PCR reaction is more than sufficient in most cases, you can test it with a postive control first. You don't have to do any dilutions as the cDNA concentration is not accurately known from the start. However, if you wish to dlute your template you can just use deionized water. To minimize degradation of your cDNA sample, avoid repeated freeze-thawing.